Environmental Performance of Xylene, Hydrochloric Acid and Ammonia Solution During Pap Stain for Diagnosing Cervical Cancer

hydrochloric acid and ammonia (5.5 liters each) over nine months, and all diagnoses coincided with the cytological details. Conclusions . The Eco-Pap is an innovative method that transforms Pap testing into a sustainable and supportable technology. Health 11: 58–65 (2016)


Introduction
In countries with a low degree of development, cervical cancer is a very frequent neoplasia, with a high number of deaths in women during a time in their lives when they play an important role in the family. 1,2,3 Cervical cancer is also a reflection of inequality and poverty, as it affects poor and rich countries differently. At least 80% of deaths caused by cervical cancer occur in countries with a low degree of development. 3,4 Peru evidences a similar inequality, with many patients with cervical abnormalities in very poor populations, mainly with HSIL (high squamous intraepithelial lesion) and carcinomas, due to low levels of healthcare and social support, and as a consequence, there are high levels of mortality for this disease. 1, 5 The most commonly used tests in cervical cancer diagnosis include the Papanicolaou (Pap) test and cervicoscopy, along with testing and genotyping for HPV, a common sexually transmitted infection and causal agent of cervical cancer. 6,7,8 Cervical cancer diagnosis requires quality, reliable results and testing methods should have a good costbenefit ratio. However, low importance has been placed on the sustainability of these methods, at a cost to global environmental health.
The conventional Pap testing method 22,23 features 17 steps, grouped in five stages (hydration, nuclear staining, cytoplasmic staining, dehydration, clearing) using 3 stains (Harris Haematoxylin, Orange G and EA). Its environmental impact may be reduced with xylene substitutes used in clearing and automation of analytical processes. However, these modifications may have a low cost-benefit ratio, may be complicated in their implementation and development, and unaffordable in countries with a low degree of development, as in Peru, where conventional techniques like colposcopy and Pap test are produced in large quantities. 9,10 The wide applicability of cytological screening using the Pap

Methods
An experimental, prospective, cross-sectional cytology study was performed in the pathological anatomy service of the diagnosis assistance department of the Hospital Nacional Docente Madre Niño "San Bartolomé" (HONADOMANI SB) in Lima, Peru. Sample size was calculated using EPIDAT 4.1 (Xunta de Galicia, España), considering a sensitivity of 0.95, a heterogeneity of 50% and a margin of error of 0.04, and obtaining a sample size of 2,500 cervical smears.
Only cervical smears that met the requirements for technique competence and normalized procedures in the manual of the Health National Institute of Peru, standard quality control measures according the Bethesda System 2014, and standardize operational procedures for each test were considered. 11,12,13 Samples were prepared in a conventional preparation and sent to the laboratory for cervicaluterine screening, among five health networks and micro-networks in five districts (Los Olivos, Rímac, San Martín de Porres, Lima and Tupac Amaru).

Analytical Procedure
The method used for screening the cervical smears was ecological modification of the Pap test, as described in Table 1 , before its final use, then pulling away the filtered fraction to gauge the preparation for the new reagent, achieving a complete recycling of the reagent. The process of filtering is performed 10 times. In the last step, the reagent is stored inside a 2 L dark container for a year, and during this time it can be reused for staining, due to the slow maturation of hematoxylin.
The second phase consists of a single alcohol bath, followed by polychromatic staining that is a combination of conventional EA-36, preferentially with conventional Orange G-6. This reduces the number of alcohol baths used in both steps for cleaning excess stain.
Lastly, in the clearing phase, two or three baths in xylene were eliminated, leading directly to mounting in anhydrous resin Entellan® or new Entellan® (Merck, Darmstadt, Germany), which contain fewer toxic substances and in lower amounts in parts-per million (ppm) -100 ppm average over an eight-hour workday and retain the same function for obtaining optimal microscopic visibility under the same optimal conditions as gold standard staining, as demonstrated photographically. 24,25

Validation Study
The method was subject to a validation study before its application to determine staining quality in comparison to the conventional gold standard Pap testing.This validation was evaluated following the manual of technique competence and proceedings of the National Institute of Health of Peru and the inter-observational results showed that the refraction index and cellular transparency were optimal. 26, 30 All cytotechnologists and cytopathologists were evaluated and certified by the National Institute of Health of Peru and had an average level of diagnosis agreement of kappa = 0.81. All had over 12 years of experience in cytopathology. 26

Quality control and statistical analysis
The quality of the smears was evaluated based on the staining quality index (SQI) and the Bethesda 2014 guide for micro and macroscopic quality control. The data analysis was performed with SPSS version 20.0 (IBM, Armonk, USA) using inferential statistics. Non-parametric tests included Cohen's kappa, and diagnostic tests and ROC curve were evaluated during the validation phase. The evaluation method was performed with 3,906 smears evaluated with both methods. All slides were analyzed by seven cytotechnologists and five cytopathologists.

Ethics
This research was approved by the Ethics and Research Committee of HONADOMANI SB.

Method Validation
Double-blind experiment validation of Eco-Pap versus conventional staining was performed in 5% of cervical smears 30 days before the beginning of implementation in HONADOMANI SB (December 2014), obtaining a Kappa correlation index of 0.89 between methods. The cytological validation results are shown in Figure  1 and 2.

Evaluation of contaminant diminution rates
The processes for reducing institutional contamination were evaluated quarterly for each eliminated toxic or carcinogenic reagent. Additionally, the quarterly savings was estimated for each reagent (Figures  3 and 4). For the 52,319 Pap tests, Eco-Pap used 66 fewer L of xylene, 5.5 fewer L of HCl, and 5.5 fewer L of NH 3 compared to the conventional Pap testing method (Figures 5 and 6). Thus, Eco-Pap significantly reduced the biocontamination hazard generated by use of toxic or carcinogenic reagents, reduced the bioaccumulation of xylene (resulting from accumulation of used xylene bottles in the cytology area) and proved to be an environmentally protective method.

Cytological valuation
The cytological staining was homogenous, and the refraction index and cellular transparency were optimal for the tridimensional cellular disposition. The sensitivity and specificity of the method was 57% and 98%, respectively.
The cellular details for diagnosis of normal and neoplastic cytology coincided with the specific details of each lesion. The characteristics for atypical squamous cells of undetermined significance (ASCUS), low grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HSIL), cervical intraepithelial neoplasia, squamous cell carcinoma and adenocarcinoma in situ were clearly appreciable (hyperchromasia, altered nucleus:cytoplasm (N:C) ratio, nuclear irregularities, etc.) (Figures 7, 8, 9, 14).

Eco-Pap cytological staining details
Research suggests the need for regional and global organizations to design manuals for better management of toxic and carcinogenic reagents in cytology laboratories.
Eco-Pap reduces the use of toxic and carcinogenic reactants and lowers acquisition costs and handling of these reactants, costs that could be distributed to other areas of institutions or to new diagnostic methods in cytology such as HPV genotyping. Furthermore, reducing the staining steps enables a faster diagnostic response (it generally takes from four to seven weeks with a loss of patients between 40 and 60%) for the treatment and monitoring of cervical cancer. The cost per Pap test could also be reduced, making it more accessible to populations with few resources in marginal urban areas that are generally far from healthcare centers. Moreover, this method makes it possible to maintain costs in periods of economic shifts, inflation and international dollarization (Figure 4).
Eco-Pap yields results that are comparable to the gold standard staining method (kappa=0.89) and has a more favorable cost-benefit ratio than commercial modifications of staining, allowing for diagnosis of cervical cancer at a lower economic cost. 11,17,18,19,28 Moreover, if every cytology laboratory in the world ceased using xylene during clearance in Pap staining, the environmental impact caused by industrial production of xylene would be greatly reduced. 29 In this study, 66 fewer liters of xylenes were used with the Eco-Pap method. A great amount of pollution could be prevented if all hospitals in the world that use this solvent would decrease its use, and the production of xylene would also be decreased. Xylene is obtained through catalytic

Discussion
Eco-Pap reduced the environmental contamination by xylene, hydrochloric acid and ammonia, achieving a reduction (prevention) of 66 L of xylene, and 5.5 L each HCl and NH 3 over three quarters, compared to what is conventionally used during Pap testing (Figure 3), and at the same time, allowed for the diagnosis of cervical malignancies (LSIL, HSIL, carcinoma in situ, adenocarcinoma in situ, etc.) (Figures 7-14), leading to environmental, social and health benefits for the community. In addition, the Eco-pap method had a total quarterly cost savings of US $11,505.00.
Eco-Pap uses progressive hematoxylin, polychromic solution and direct mounting, which eliminates and reduces the use of environmentally toxic and carcinogenic reagents, reducing the impact of occupational exposure, given that adequate protection materials are not routinely used in cytology laboratories in Peru. 14,15,16,29 Inhalation of xylene can cause irritation in the mucous membranes of the nose and throat, and in high concentrations can produce nausea, vomiting, headache, serious respiratory difficulties, cough, heart anomalies, proteinuria and hematuria after excessive inhalation. 29, 31 It can induce neurological disorders, and xylene and hydrochloric acid are associated with the development of cancer, leukemia and lymphocytic brain tumors in cases of chronic exposure to high concentrations over long periods of time. 32 Similarly, hydrochloric acid and ammonia produce intoxication and environmental problems due to the pollution they produce. 33 Prolonged exposure to both reagents, both by direct contact and inhalation, can be damaging to the health of people exposed to them. 34 Manifestations of intoxication by these reagents are heterogeneous, but can affect the respiratory system (cough, dyspnea, wheezing, bronchopulmonary problems), the nervous system (stupor, confusion, lack of coordination, difficulty in walking), skin and mucous membranes (burns, glottis edema, sight loss, temporary blindness), and the circulatory system (weak and rapid pulse, fever, decrease in blood pressure), among others. 16 The Eco-Pap is a sustainable and supportable method that allows for the effective diagnosis of cervical cancer, and we propose that the name 'Eco-Pap' may be attractive to clinicians and patients.

Conclusions
Cervical cancer is an important public health issue and is associated with high morbidity and mortality in underdeveloped countries. It is primarily diagnosed by the use of the Papanicolaou test which employs highly polluting toxic and carcinogenic reagents (xylene, hydrochloric acid and ammonia), generating a harmful work environment depending on the duration of exposure.
In spite of the advent of commercial modifications of conventional Papanicolaou testing, current methods have a low cost-benefit ratio and are unable to be routinely used in the diagnosis systems of the healthcare centers in Lima and Peru. Our proposed Eco-Pap method eliminates the use of environmentally toxic and carcinogenic reagents, reduces the risks due to occupational exposure and has a better cost-benefit ratio for patients.
The Eco-Pap is an innovative, sustainable and supportable technology.